Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

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Ten 15‐m3 outdoor ponds were treated daily for 8 weeks with a synthetic coal‐derived crude oil; ecological effects were monitored for an additional 52 weeks. The experimental design included two replicate ponds at each of five oil input rates (from 1 to 16 ml/m3/d) plus two untreated controls. A gradient of responses was observed across the gradient of treatment levels. Cladoceran zooplankton populations and ecosystem metabolism (production/respiration) were affected at the lowest input rate, but the effects disappeared before the end of the oiling period and this exposure level (approximately 3% of the 48‐h LC50 for Daphnia magna) was considered safe for this ecosystem. At the next higher treatment level, effects on zooplankton and ecosystem metabolism were greater and persisted until the oiling ended; reproduction of mosquitofish (Gambusia affmis) was also impaired. Major changes occurred throughout the ecosystem at higher treatment levels. The two highest treatment levels completely disrupted the pond community: The ponds recovered from the next‐to‐highest treatment but the effects of the highest treatment persisted for more than a year. Indirect effects occurred at all treatment levels and included changes in water quality, replacement of sensitive taxa by more tolerant competitors and changes in abundance of some species because of increases or decreases in their predators. The results of this experiment were qualitatively and quantitatively similar to those of a parallel experiment in pond‐derived microcosms, and thus substantiated the ability of the microcosms to simulate larger, more natural ecosystems.

In acute toxicity tests, green algae Selenastrum capricornut'um, diatoms Nitzschia palea, adult snails Physa gyrina, juvenile cladocerans Daphnia magna, larval midges Chironomus tentans, adult amphipods Gaintaurus minus, juvenile fathead minnows Pimephales promelas, and embryo-larva stages of rainbow trout Salmo gairdneri and largemouth bass Micropterus salmoides were exposed for 4 hours (algae), 48 hours (arthropods and snails), 96 hours (fathead minnows), 7 days (largemouth bass), and 27 days (rainbow trout) to two phenols (phenol and t%naphthol), two azaarenes (quinoline and acridine), and two polycyclic aromatic hydrocarbons (naphthalene and phenanthrene) present in coal-derived oils. Median lethal or median effective concentrations (LC50s or EC50s) ranged from 0.03 mg/liter for phenanthrene and rainbow trout to 286.54 mg/liter for phenol and the green alga. The rainbow trout embryo-larva assay was the most sensitive of the test systems to all the chemicals except quinoline. For this last compound, systems with juvenile fathead minnows and largemouth bass embryos were the most sensitive. As test systems, fish embryos and larvae were the most sensitive, juvenile fathead minnows and arthropods had intermediate sensitivity, and algae and snails were the most resistant to the test compounds under the test conditions. Within each chemical class (phenols, azaarenes, and polycyclic aromatic hydrocarbons), toxicity increased with increased ring number except for the reversed relationship with the azaarenes and fathead minnows. Thus, tJ-naphthol (two rings) was 2 to 45 times more toxic than phenol (one ring); acridine (three rings) was 7 to 27 times more toxic than quinoline (two rings); and phenanthrene (three rings) was 3 to 9 times more toxic than naphthalene (two rings). There was also a relationship between increases in toxicity and increases in the calculated octanol-water partition coefficients of the compounds.